JPH01104087A - Production of immunosuppressive substance - Google Patents

Abstract

PURPOSE:To obtain an immunosuppressive substance having excellently immunosuppressive action, even developing no excessive cytotoxicity, from a culture mixture obtained by cultivating a fungus belonging to the genus Isaria, capable of producing an immunosuppressive substance. CONSTITUTION:First, a fungus [e.g. Isaria sinclairii (ATCC No.24400)] belonging to the genus Isaria is cultivated preferably by aerobic submerged culture. Then an immunosuppressive substance is collected from the culture mixture by a well-known method. The culture temperature is preferably 25-30 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a new method for producing immunosuppressive substances, particularly myriocin, using microorganisms.

[Conventional technology]

It has been known that immunosuppressive substances derived from microorganisms can be produced by culturing immunosuppressive substance-producing strains belonging to the genus Cylindrocarpon, Tolypocadium, or Fuserrum. In particular, cyclosporine produced from the genus Tribofragilum is widely used to prevent rejection during organ transplants.

[Purpose of the invention]

In view of these circumstances, the present inventors searched for immunosuppressive substance-producing bacteria, and as a result, they discovered immunosuppressive substance-producing bacteria from microorganisms belonging to a certain genus that was not previously known to produce immunosuppressive substances. They discovered this and arrived at the present invention.

[Structure of the invention]

That is, the gist of the present invention is IsarIa
The present invention relates to a method for producing an immunosuppressive substance, which comprises culturing an immunosuppressive substance-producing bacterium belonging to the genus and collecting the immunosuppressive substance from the culture.

The present invention will be explained in detail below.

The microorganism used in the method of the present invention belongs to the genus Isaria and is an immunosuppressant-producing bacterium that has the ability to produce a sufficient amount of immunosuppressant in culture.

Examples of such strains include 5 inclairii, which belongs to the genus Isaria, and is listed in the American Type Culture Collection.
11ection) to l5aria 5inclai
rii ATCC No. 24400.

The immunosuppressive substance-producing bacteria used in the present invention are not limited to these strains, and of course include mutant strains that have been modified by artificial mutation means such as commonly used ultraviolet rays, high-frequency radiation, and chemicals.

The immunosuppressant-producing bacteria used in the present invention can be cultured in various culture media containing common nutrient sources for molds.

For example, carbon sources include glucose, starch, glycerin, sugar syrup, dextrin, molasses, maltose, etc., and nitrogen sources include cornstarch liquor, peptone,
These include yeast extract, potato broth, meat juice, soybean flour, wheat germ, potassium nitrate, sodium nitrate, ammonium sulfate, amino acids, and other common inorganic salts and organic salts that aid surface development and promote the production of immunosuppressive substances. Additives commonly used in culture, such as inorganic substances and antifoaming agents, can be appropriately added.

Although the culture method is not particularly limited, an aerobic deep culture method is suitable. The appropriate temperature for culturing is 20-35℃.
, preferably at 25-30°C.

The immunosuppressive substance produced in the culture by the method of the present invention can be extracted from the culture by appropriately combining commonly used operations such as extraction and adsorption as necessary. For example, insoluble substances such as bacterial cells are separated from the culture solution by filtration, centrifugation, etc., and the culture filtrate is passed through Amberlite XAD-2 to adsorb the immunosuppressive substance. The immunosuppressive substance thus obtained may be further treated with, for example,
A highly purified product of the immunosuppressive substance can be obtained by elution with methanol and subsequent fractionation of the eluate by reverse phase chromatography.

The highly purified immunosuppressive substance obtained by
syces ) and Mycelia sterilia (Myc
Myriocin, also known as thermofimosidin (Ther+IIopy+wocidin), which is also produced by bacteria such as Elia 5terilia)
C (see U.S. Pat. No. 3,928,572)] was estimated from NMR, ultraviolet-ultraviolet absorption spectrum, infrared absorption spectrum, mass analysis, etc. This substance also has strong immunosuppressive activity.

[Effect]

The immunosuppressive substance produced by the present invention exhibits excellent immunosuppressive effects and is used as an agent for suppressing rejection reactions during organ or bone marrow transplantation in mammals such as humans, cows, horses, dogs, mice, and rats. As a prophylactic or therapeutic agent for autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, allergies, or diseases based on abnormal lymphocyte proliferation.
Alternatively, it can be used as a reagent in medicine and pharmacy.

When the immunosuppressive substance of the present invention is used as the above-mentioned medicine, it can be mixed with carriers, excipients, diluents, etc., formulated into powders, capsules, tablets, injections, etc., and administered to patients. Alternatively, it may be freeze-dried.

The dosage may vary depending on the disease, symptoms, weight, sex, age, etc., but for example, to suppress rejection in kidney transplantation, it is usually 0.1 to 10 μg/day (titer) for adults.
is administered in one to several divided doses a day.

〔Example〕

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way by the following examples unless the gist of the invention is exceeded.

The activity of the immunosuppressive substance was measured by the following method.

Various immune reactions using mouse, rat, or human lymphocytes can be used to measure the activity of the immunosuppressive substance. For example, the immunosuppressive substance of the present invention
Allogeneic lymphocyte mixed reaction of mouse, rat, and human (allogeneic M
Allogeneic MLR refers to lymphocytes from two individuals of the same species but with different major histocompatibility antigens, such as lung cells, lymph node cells, peripheral blood lymphocytes, etc. This is a lymphocyte blastogenesis reaction induced by mixed culture of this allogeneic MLR.
This is a phenomenon that reflects and is induced by differences in major tissue compatibility antigens between lymphocyte donors. No, so allogeneic MLR is, for example, a widely used method for donor-recipient selection in organ transplants.

Normally, when performing allogeneic MLR, one lymphocyte is used as a stimulator cell after its division and proliferation is inhibited by X-ray irradiation or mitomycin C treatment, and the other lymphocyte (responsive cell) is immature. A method for measuring rejuvenation reactions (one say-MLR") can be used.

Furthermore, the activity of the immunosuppressive substance of the present invention can also be measured as the activity of suppressing the induction of cytotoxic T cells having major histocompatibility antigen-restricted properties induced during allogeneic MLR.

In addition, the activity of the immunosuppressive substance of the present invention suppresses the blastogenic response of lymphocytes induced by stimulation with various mitogens (concanavalin A1 phytohemagglutinin, Hawklide mitogen, etc.) in addition to allogeneic MLR. or lymphocytes induced by cytokines (such as interleukin 1.2.3.4.5.6) that have the activity of enhancing the division and proliferation or promoting differentiation of lymphocytes such as T cells and B cells. It can also be evaluated as an activity to suppress the division/proliferation response of spheres or the inducing activity of functions. Furthermore, it is possible to evaluate the activity of suppressing the production of these cytokines from T cells, macrophages, etc.

Furthermore, the immunosuppressive substance of the present invention can be administered intraperitoneally to mice, etc.
Activity of suppressing the induction of plasma cells that produce anti-xeno-erythrocyte antibodies induced in the tile cells of mice previously immunized with xeno-erythrocytes, etc., by oral, intravenous or subcutaneous administration, or allogeneic mice. It can also be evaluated for its activity in suppressing the graft-versus-borrower reaction caused by skin transplantation, delayed-type allergies, adjuvant arthritis, etc.

In addition, MRL/fp, a mouse model of autoimmune disease,
By administering the immunosuppressive substance of the present invention to r+7 mice, NZB/WF mice, BXSB mice, etc., suppressive activities such as anti-DNA antibody production, rheumatoid factor production, nephritis, lymphocyte proliferation abnormalities, etc. It can also be evaluated as a life-prolonging effect.

〔Example〕

Example 1 (Jar culture of Sinclairie strain) GP
Y medium (per 12, glycose 30g.

Peptone 5g, yeast extract 3g, Kl, POa
O, 3g, KZHPOa 0.3g, MgSO4・7
HR00.3g, pH5.5) to 500Ild! Dispense 100 d into two shake flasks and add 121°C.
Isaria sinclairi AT grown on potato dextrose agar after sterilization by autoclaving for 20 minutes.
Approximately 1 c4 of mycelium of CC No. 24400 was inoculated into each flask, and cultured at 25°C for 6 days with reciprocating shaking at 4% (145 rpm).
pmS shaking width 8 cm). The obtained culture solution was used as a seed mother to inoculate into a 10ffi-capacity Jafermentor containing 5 Il of the above-mentioned GPY medium.
℃, 10 days aerated agitation culture (IVVM, 300 rpm)
) was carried out.

Example 2 (Collection of immunosuppressive substances from the culture solution of Bacillus cinclairii) The culture solution obtained in Example 1 4.5j! The bacterial cells and insoluble matter were removed by filtration to obtain a culture filtrate 4.02. The obtained culture filtrate was transferred to Amberlite XAD-2 (φ40+m
The solution was passed through a 5×750 tube to adsorb the immunosuppressive substance.

Further, water 41 was passed therethrough for washing.

Thereafter, methanol 61 was passed through the tube to elute the immunosuppressive substance. This was concentrated under reduced pressure, dissolved in 200 ml of ethyl acetate, and extracted three times with 200 ml of water using a separatory funnel.

The water extracted part and the ethyl acetate part were each concentrated under reduced pressure and lyophilized to give 2.23 g of immunosuppressant 1 and 0.34 g.
g was obtained.

Example 3 (Collection of myriocin) Immunosuppressant obtained by freeze-drying the water extract obtained in Example 2! Dissolve 2.23 g in 5 d of water and apply it to a reverse phase chromatography (ODS DM-10207 manufactured by Fuji Davison Chemical Co., Ltd.) column (φ 30 mm x h 8
Elution was started with water and fractionated by elution while gradually increasing the methanol concentration. 70%
After concentrating the methanol eluted portion to dryness under reduced pressure, it was dissolved in a small amount of hot methanol and left to cool to precipitate myliosin crystals.The precipitated crystals (0) were dissolved again in hot methanol and recrystallized to obtain pure myliosin 40. I got ■.

[Experiment example]

Experimental Example 1 (Measurement of immunosuppressive effect of collected immunosuppressive substance) The activity of the immunosuppressive substance of the present invention was measured using mouse allogeneic lymphocyte mixed reaction (hereinafter sometimes referred to as MLR). Mouse allogeneic MLR uses BAL B/c mouse (H-2-) tile cells as reactor cells and C57BL/6 (H-2') mouse tile cells treated with mitomycin C as stimulator cells. , by mixed culture in equal ratios.

The reaction cells were prepared by the following method. 5
The lungs were removed from ~6-week-old male BALB/c mice and placed in RPM11640 medium (hereinafter sometimes referred to as Fe2) supplemented with 5% heat-inactivated fetal bovine serum (hereinafter sometimes referred to as Fe2).
Kanamycin sulfate 60μg/rrll, L-glutamine 2mM 5N-2-hydroxyethylpiperazine-N'-
2-ethanesulfonate (HEPES) 10mM, 0.
(containing 1% sodium bicarbonate) to obtain a single cell suspension of tile cells.

After the exsanguination process, using RPMI1640 medium containing 10-'M2-mercaptoethanol and 20% FC3,
The cells were prepared at 10'' cells/day and used as a reaction cell suspension.

The stimulated cells were prepared as follows. Lungs were removed from 5-6 week old male C57BL/6 mice and RPM
A single cell suspension of tile cells was obtained using 11640 medium.

After hemolysis treatment, 40 μg/d mitomycin C at 37°C.
The treatment was carried out for 60 minutes. After 3 washes, 10-'M2
- RP containing mercaptoethanol and 20% FC3
Using M11640 medium, prepare 107 cells/#2e and use as a stimulated cell suspension.

Add 50 μl of the reactive cell suspension prepared by the method described above, 50 μl of the stimulated cell suspension, and 100 μl of the test body fluid to a 966 micro test plate, and add 50 μl of the reaction cell suspension prepared by the above method to a 966 micro test plate,
Culture was performed for 4 days under carbon dioxide gas conditions.

MTT (
C3(4,5-dia+ethylthiazol-2
-yl)-2,5-diphenyltetrazol
A dye quantitative method using .

1) Pigment assay method using MTT After completion of culture, remove 100 μl of supernatant from each well,
mg/d MTT solution at 20a1. each of the cells was added to each -all and incubated at 37°C for 4 hours. Thereafter, 100% of a 0.01N hydrochloric acid solution containing 10% sodium dodecyl sulfate was added. Add N and incubate overnight at 37°C to dissolve the purple formazan crystals that formed.
The absorbance at 550 nm was measured using a microplate spectrophotometer, and was used as an index of the lymphocyte rejuvenation reaction of mouse allogeneic MLR.

Suppression of mouse allogeneic MLR was evaluated by calculating the suppression rate using the following formula.

2) Method using 3H thymidine incorporation as an indicator After the completion of culture, 0.5 μCi/we11 of 1H thymidine was added,
After culturing for a period of time, the cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter, which was used as an indicator of lymphocyte development in mouse allogeneic MLR. Suppression of mouse allogeneic MLR was evaluated by calculating the suppression rate using the following formula.

[Margins below] The immunosuppressive substance of the present invention is soaked in methanol, then RPM
Diluted in 11640 medium, 10 and 1 μg/rd
was used at a final concentration of Note that methanol was used at a final concentration of 0.1% or less, and in this case, no influence was observed on the homogeneous MLR.

For the immunosuppressive substances of the present invention (water extract fraction and ethyl acetate extract fraction), from 10 μg/ml to 10 −′μ
Mouse allogeneic ML at concentrations at the end of the range of g/rtdl
As a result of measuring the inhibitory activity of the lymphocyte rejuvenation reaction in R, as shown in Table 1, the concentration (ICso) of the immunosuppressive substance of the present invention (ethyl acetate extraction) that inhibits the mouse homologous MLR by 50%. is 1.6 xlo-'μg/ml, and the ICs of the immunosuppressive substance in water extraction. is 2.5×10-1
It was found that the concentration was μg/ml.

On the other hand, these immunosuppressive substances showed no cytotoxicity against mouse L929IEl cells, etc., even at a concentration of 10IIg/id (IC3°: 50μg7'ml [margin below]) Experimental Example 2 (Measurement of the immunosuppressive effect of myliocin) ) The immunosuppressive effect of myliocin was measured using 3H-thymidine incorporation as an indicator in the same manner as in Example 1, and the results are shown in Table 2.Myliocin was suspended in methanol. The final concentration of is 0.05%
Used below, in this case no effect on homologous MLR was observed.

IC of myriocin against mouse homologous MLR, based on the results shown in Table 2. The value is 10-
``It became clear that:

[Left below] Experimental example: ((Suppressing effect on mouse tile cell blastogenesis response due to mitogen stimulation) Effect of phytohemagglutinin (PHA) or Hawkelyde mitogen (PWM) stimulation on mouse tile cell blastogenesis response. The test was conducted in the following manner.

The lungs were removed from 5-8 week old male BALB/c mice and RPM was added with 5% heat-inactivated beef tallow serum.
A single cell suspension of lung cells was obtained using 11640 medium.

After hemolysis, the concentration was adjusted to 5xlO'/d using RPM11640 medium containing 10-'M2-mercaptoethanol and 20% 0% heat-inactivated beef fat serum, and PHA or PWM
was added. Add 100 μl of this cell suspension to each well of a 966 micro test plate containing 100 μl of test solution.The number of mouse tile cells is 5x1.
After culturing for 72 hours at 37°C and 5% carbon dioxide, 0.5 μCi/well of 3H-thymidine was added.
Add well 1. The cells were cultured for an additional 4 hours under the same conditions. After culturing, the cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter, which was used as an indicator of the mouse tile cell rejuvenation reaction. The results are shown in Table 3.

[Margin below]

As is clear from Table 3, the immunosuppressive substance of the present invention is more effective than l-'HA or PWM compared to the control that does not contain the same substance.
311-thymidine incorporation induced by 311-thymidine was strongly suppressed.

Experimental Example 4 (Rat lung cell concanaba JA (Con
A) Suppressive effect on 3H-thymidine uptake of IL2-dependent mouse cell line CTLL-2 induced by interleukin 2 (IL2) in stimulated culture supernatant) IL2-dependent mouse cell line CTLL-2 was mixed with 30% beef fat 2 x 10S cells/d in RPM11640 medium containing serum
It was prepared as follows. 50 μl of this cell suspension and 50 μl of rat tile cell Con A stimulated culture supernatant containing IL2 were added to each well of a 966 microtest plate in which 100 μ2 of the test solution had been previously placed. After culturing for 20.44 and 688 hours at 37°C and 5% carbon dioxide, 3H
- 0.5 μCi/well of thymidine was added, and the cells were further cultured for 4 hours under the same conditions. After the culture was completed, the cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter. The results are shown in Table 4.

space below] As is clear from Table 4, the immunosuppressive substance of the present invention is
The increase in 3H-thymidine uptake in CTLL-2 cells induced by IL2 was strongly suppressed.

Experimental Example 5 (Suppressive effect on IL2 production in mouse allogeneic mixed lymphocyte culture (MLC)) Mouse allogeneic MLC was carried out as follows. That is, 0.5 Id each of the reacted cell suspension and the stimulated cell suspension prepared in the same manner as in Experimental Example 1 were added to a 24-well multi-dish containing test solution 1I11 in advance, and incubated at 37°C for 5 % carbon dioxide gas condition for 3 days. After completion of the culture, the supernatant was collected and used as a supernatant of mouse allogeneic MLC.

IL2 activity in the supernatant of mouse allogeneic MLC was measured as follows. That is, IL2-dependent mouse cell line C
RPM containing 30% heat-inactivated live fetal serum for TLL-2
I1640 medium was prepared at 10' cells/Id, and 100 μl of the above MLC supernatant was added in advance.
100 pl was added to each well of a six-micro test plate. After culturing for 20 hours at 37°C and 5% carbon dioxide, 0.5 μCi/well of aH-thymidine was added.
The cells were further cultured for 4 hours under the same conditions. After the culture was completed, the cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter, which was used as an index of 1L2 activity. The results are shown in Table 5.

[Margin below]

As shown in Table 5, the immunosuppressive substance of the present invention has t
Suppresses IL2 production in mouse allogeneic MLC
°(It was suggested that
Experimental Example 6 (Mouse allogeneic lymphocyte mixed culture (M
Suppressive effect on allogeneic cytotoxic T cells induced by LC) /) 2 Experimental Example 1
BALB/1c mouse (H-2') lung cell suspension (2xlo'b) prepared in the same manner as
cells/ml), 0.5 ml of mitomycin C-treated Ct+157 B L/6 mouse (H-2') lung cell suspension (2X 10' cells/ml) and 1.0 relatives of the subject. In addition to 24-hole multi-date, 37°C
Culture was carried out for 6 days under 5% carbon dioxide conditions.

After the completion of the culture, the cells were collected by centrifugation and placed in 5×10” RPM [1640 medium containing 10% FC3].
m to 6.25 x 10' cells/Id and used as effective cells. ! : As target cells, C57B L/b mouse-derived leukensis cells Ld that are syngeneic (H-2'')- with the stimulator cells.
used force. lO″ EL4 cells were injected with 100 μCi of J
a! ” Using Cr0n (1mc i/Id), 37
SIC by incubating for 1 hour at :.

After incorporation into the cytoplasm, wash and 104 cells
rl and used as target cells.

Measurement of cytotoxic activity was performed using 0.1 d of effective cell suspension;
Add 0.1 ml of target cell suspension to a 96-well flat bottom plate and culture at 37°C for 4 hours. Measure the amount of Cr released in the supernatant, and calculate cell damage using the following formula: Activity was calculated.

In addition, the cytotoxic T-cells induced by the above method are
Stimulator cells (H-2') and syngeneic EL4 cells (H-2'
) showed strong cytotoxic activity7, but against allogeneic Me
Since the cells showed no cytotoxic activity against thA cells (H-2'), it was suggested that they were H-2''-restricted allogeneic cytotoxic T cells.

As a result of adding the immunosuppressive substance of the present invention at a concentration of 0.01 to 1 μg/ml and examining the effect on induction of allogeneic cytotoxic T cells, as shown in Table 6, almost no cytotoxic activity was observed. With the immunosuppressive substance of the present invention,
Induction of allogeneic cytotoxic T cells was significantly suppressed.

[Margin below]

Experimental Example 7 (Suppressive effect on proliferation of IL3-dependent mouse cell line FDCP2 induced by interleukin-3 (IL3)) IL3-dependent mouse cell line FDCP2 was grown at 2 x 105 cells in RPMI1640 medium containing 10% tallow serum. /d. 100 μl of this cell suspension and 50 μl of the culture supernatant of mouse leukemia cells WEHI3 containing IL3 were added to 100 μl of the test sample in advance. The cells were added to each well of a 96-well microtest plate in which the cells had been placed, and cultured at 37°C under 5% carbon dioxide gas for 20 hours. After completion of culture, iH
- After adding 0.5μC4/well of thymidine and further culturing under the same conditions for 4 hours, the cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter. It was used as an indicator of dependent proliferation.

As shown in Table 7, the immunosuppressive substance of the present invention
The immunosuppressant of the present invention suppresses the increase in 5) l-thymidine uptake of FDCP 2 cells induced by 3 (approximately 60% at a concentration of 1 μg/d). It was suggested that the substance was present.

Table 7 ---1719±439- + , --16211±629-+ Immunosuppressant 10 2179±197 96.8+
Immunosuppressant 1 7082±350 63.0
+ Immunosuppressant 0.1 11526±150
32.3+ Immunosuppressant 0.01 1183
8±378 30.2 (In the table, "+" means the presence of IL3, and "-" means the absence of IL3.) Experimental Example 8 (Interleukin 1 (IL) in mouse leg line cells
I) Inhibitory effect on response) The thymus was removed from a 7-week-old male C3H/H□N mouse, and a single cell suspension was prepared using serum-free RPM11640 medium. After washing three times with the same medium, 20% fetal bovine serum sx
lO-'M/2-mercaptoethanol, 2XIO-3
M/l, -glutamine, +xio-'M sodium pyruvate phytohemagglutinin (PHA, Wellcome Inc., HA16/17) 1 Hg/d and human ultrapear interleukin 1 (Genzyme Inc. GUPi-
1) 1.5 in RPM11640 medium containing 2 units/d
were suspended at a concentration of X10-' cells/mfl. 100 μl of this cell suspension and 100 μl of a solution containing the immunosuppressive substance of the present invention or cyclosporin A were mixed in each well of a 96-well flat-bottomed microtest plate.
After culturing for 66 hours at 15% carbon dioxide at °C,
- Thymidine was added at 0.5 μC4/well and cultured for further 6 hours. After the culture was completed, the cells in each well were collected on a filter using a multiple cell Burhester, and the radioactivity incorporated into the cells was measured by a liquid scintillation method using a toluene-based scintillator.

The results obtained by the above method are shown in Table 8.

In the table, SD represents standard deviation, and the inhibition rate (%) was calculated using the following formula.

Suppression rate (%) = Table 8--1389± 42- PHA -3270± 316-PI
LI 1180
3 ± 1740 0 ÷ immunosuppressant 2 3973 ± 39 91.80.2
4646± 826 83.9 As shown in Table 8, the immunosuppressive substance of the present invention suppresses charcoal-dependent ILI
It showed a response suppressing effect.

Experimental Example 9 (Interleukin 6 (IL) in mouse lung cells
6) Suppressive effect on response) Lungs were removed from male 8-week-old BAL B/c mice, a single cell suspension was obtained using serum-free RPM11640 medium, and the cells were separated by centrifugation (1000 rpm, 5 minutes). was formed. To this was added 0.16 M ammonium chloride solution and 0.17 M Tris solution (pH 7,65).
The red blood cells were lysed by adding solutions mixed at a ratio of 1 to 1, followed by 3 ml of serum-free RP MI 1640 medium.
Washed twice. The obtained cells were added with 20% beef tallow serum, 5X
10-'M/2-mercaptoethanol, 2x 10
-3M/L-glutamini/lXl0-"M sodium pyruvate and 7 as a source of interleukin-6
RP M containing 25% human bladder cancer cell line culture supernatant
11640 medium at a concentration of 5XIO'm cells/d. 100μ2 of this cell suspension and 100μ2 of a solution containing the immunosuppressive substance of the present invention or cyclosporin A were mixed in each well of a 96-well flat-bottomed microtest plate, and the mixture was kept at 37'C and 5% carbon dioxide for 72 hours. Cultured.

After culturing, 50με of the cell suspension was collected from each well, 300μ of RPM f1640 medium containing 0.7% agarose gel, 20μ of physiological saline containing 1140% protein A-bound sheep red blood cells. and 20 μl of anti-mouse IgG antiserum diluted 300 times with physiological saline were mixed in a test tube and spread uniformly on a Rodac plate (Falcon 1034). After the gel was left at room temperature for several minutes to harden, it was incubated at 37°C under 15% carbon dioxide gas conditions for about 4 hours.

300 ul of guinea pig complement diluted 40 times with serum-free RPMT1640 medium was added to each plate, and after further incubation for 2 hours, the number of plaques formed was counted under a stereomicroscope.

The results obtained by the above method are shown in Table 9.

In the table, SD represents standard error, and the inhibition rate (%) was calculated using the following formula.

As shown in Table 9, the immunosuppressant of the present invention exhibited a concentration-dependent 11,6 response suppression effect.

Experimental Example 10 (Suppressive effect on anti-sheep red blood cell antibody production in mice) After immunizing male 7- to 8-week-old BALB/c mice with sheep red blood cells (SRBC), the lungs were removed on the 3rd or 4th day. By measuring the number of plaque-forming cells (PFC), the inhibitory effect on anti-sheep red blood cell antibody production was examined as follows.

Experiment 1: The immunosuppressive substance of the present invention was continuously administered for 4 days from 3 days before the immunization day to the day of immunization, and then 5R
The mice were immunized with BC (1xlO' cells/mouse), and administration was continued for 3 days starting from the next day. On the fourth day after immunization (the day after the final administration), the lungs were removed and the number of anti-sheep erythrocyte antibody producing cells was calculated by direct plaque method. At this time, the mouse body weight, wet weight of thymus and lung, number of lung cells, and blood urea nitrogen (BUN) value were also measured.

Experiment 2: The immunosuppressive substance of the present invention was administered twice at a dose of 2 mg/kg on the day before and the next day of immunization, and
Xl0'/mouse), the lungs were removed on the third day (two days after the final administration), and the number of anti-sheep erythrocyte antibody producing cells was calculated by direct plaque method. At this time, the mouse body weight, wet weight of thymus and lung, and number of lung cells were also measured. Also - for comparison, cyclosporin A (CsA
) were administered on the same schedule and similar measurements were made - the results obtained in experiments 1 and 2 are shown in the table below.

(Left below) As shown in Table 10, the immunosuppressive substance of the present invention showed anti-sheep erythrocyte antibodies in both continuous administration for 7 days (Experiment 1) and twice administration before and after the day of immunization (Experiment 2). It showed an inhibitory effect on production, that is, an effect of reducing the number of PFC per unit number of lung cells (IXIO' cells) and the number of PFC per total number of lung cells. Furthermore, the number of PFC per unit number of lung cells after two administrations was strong.

The immunosuppressive substance of the present invention had almost no effect on body weight, lung wet weight, lung cell number, and BUN value, except for a slight decrease in leg line wet weight in Experiment 2. On the other hand, Cyclosporin A has a
It showed the effect of reducing IJI viscera and lung cell numbers.

Experimental Example 11 (Cytotoxicity to mouse 929 cells) Cytotoxicity to mouse 929 cells was investigated as follows.

L929 cells were treated with F12 containing 10% heat-inactivated tallow serum.
Prepare 1.5 x 10 cells/ml with culture medium, add 100μ2 each to a 96-well microtest plate, and incubate at 37°C.
% After culturing for 24 hours under carbon dioxide conditions, the test solution 100%
μ2 was added and cultured for an additional 48 hours. It has been reported that when 100 μl of the supernatant was removed after culturing and incubated with living cells, it reacted with mitochondrial succinate dehydrogenase to produce a dark blue formazan product (J. Iwmunol, Methods).
, vol. 65, 55 Shin, 1983).

3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT, 5
mg/relative) and 20 ul were added and reacted for 4 hours at 37°C under 15% carbon dioxide gas conditions to form formazan crystals. O
Add 100μi of 1N hydrochloric acid solution to dissolve the crystals,
The reaction mixture was allowed to stand overnight at 7°C. After the reaction was completed, 100 μl of the supernatant from each well was transferred to a microtest plate for measuring absorbance, and the absorbance at 550 nm was measured using a spectrophotometer for microtest plates with 660 nm as a reference. The inhibition rate was calculated using the following formula and used as an index of cytotoxicity.

The results are shown in Table 11.

Table 11 As is clear from Table 11, the cytotoxicity of the immunosuppressive substance of the present invention to mouse 929 cells was 10 μg/d.
The following were not recognized at all.

Experimental Example 12 (Study of cytotoxicity against various cultured tumor cell lines) Cytotoxicity against various human-derived cultured tumor cell lines was examined as follows.

562, MOLT4, U9 in human-derived cultured tumor cell lines
37, HL60, KATOI [[, KB, PC-6, P
C-14 and CCRF-CEM were each grown at 2x10' cells/in RPMII640 medium containing 20% tallow serum.
Prepared in d. 50 μl of this cell suspension was added to each well of a 966 microtest plate in which 50 μl of the test solution had been previously placed. The cells were cultured for 72 hours at 37° C. under 5% carbon dioxide gas conditions, 20 ttl of a 5 μ/d MTT solution was added, and the cells were further cultured under the same conditions for 4 hours. The generated formazan crystals were mixed with 10% sodium dodecyl sulfate.
．． 100 μl of 01' normal hydrochloric acid solution was added and allowed to react overnight at 37° C. After completion of the 0 reaction, the absorbance of the supernatant of each well was measured in the same manner as in Experimental Example 4. The first part showing the results
As is clear from Table 2, the immunosuppressant of the present invention has weak cytotoxicity against various human-derived cultured tumor cell lines.
The concentration giving 50% inhibition (r csa) is 10ttg
/d or more.

[Margin below]

Table 12 (μg7ml> (%) K562 0 0.990 -0.1
, 0.886 10.6 1.0 0.905 B, 6 10.0 0.929 6.2 100.0 0.583 41. IMOL74
0 0.618 -0.1 0.560
9.4 1.0 0.559 9.5 10.0 0.499 19.3 100.0 0.074 88.011937
0 0.642 -0.1 0.619
3.6 1.0 0.567 11.7 10.0 0.497 22.6 100.0 0.156 75.7HL60
0 0.631 -0.1 0.402
36.3 1.0 0.390 38.2 10.0 0.377 40.3 100.0 0.101 84-. 0KATOII
O0,961-0,010,9900 0,10,9720 1,00,9610 10,00,8699,6 100,00,04195,7 (Continuation of 12th'A) KB 0 0.888 -0.0
1 0.898 0 0.1 G, 865 2.6 1.0 0.879 1.0 PC-600,272- 0,010,24410,3 0.1G, 245 9.9 1.0 0.209 23.2 10.0 G, 202 25.7100.0
G, 000 100.0PC-1400,787- 0,010,8010 0,10,7702,2 1,00,7327,0 10,00,7514,6 100,00,0?4 90.6 CCRF-CEM 0 0.70B
-0,010,6784,2 0,10,7090 1,00,6606,8 10,00,63710,0 100,00,01398,2 [Effect of the invention] It is clear from the description of the specification including the above examples. As shown above, substances belonging to the genus Isaria, which were not previously known to produce immunosuppressive substances, have been discovered to have excellent immunosuppressive effects, yet do not exhibit significant cytotoxicity, especially myriocin. produced.

Claims (3)

[Claims] (1) A method for producing an immunosuppressive substance, which comprises culturing an immunosuppressive substance-producing bacterium belonging to the genus Isaria and collecting the immunosuppressive substance from the culture. (2) Claim (1) that the immunosuppressive substance is myriocin
Method for producing the immunosuppressive substance described. (3) The method for producing an immunosuppressive substance according to claim (1) or (2), wherein the immunosuppressive substance-producing bacterium is Isaria cinclairii.